Method of making a gradient gel with a movable dispensing tip

Details Method of making a gradient gel with a movable dispensing tip Method of making a gradient gel with a movable dispensing tip

2001-05-03

2002-12-03

Ortiz, Angela (Department: 1732)

Plastic and nonmetallic article shaping or treating: processes

Mechanical shaping or molding to form or reform shaped article

To produce composite, plural part or multilayered article

C264S299000, C264S496000, C204S619000, C204S620000

Reexamination Certificate

active

06488880

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention comprises an apparatus and method for making a gradient gel, and more particularly, the present invention relates to an apparatus and method of using a movable dispensing device to form a uniform linear gradient across a wide gel that provides more than twenty sample lanes so that more than forty samples can be analyzed simultaneously with a conventional dual-gel electrophoretic chamber.
2. Background Art
DESCRIPTION OF THE PRIOR ART
Sixty to seventy five percent of the cholesterol in blood is associated with low density lipoproteins (“LDL”) which consist of a non-homogeneous mixture of spherical particles ranging widely in particle size (23-28 nm), buoyant density and chemical composition. Using a non-denaturing 2-16% polyacrylamide gradient gel electrophoresis, researchers have noted that individuals with a high-risk lipid profile were most likely to have primarily small, dense LDL particles, as discussed in the paper “Genetic control of low density lipoprotein subclasses,” Austin et al., Lancet 2: 592-595(3)(1986). In a case-control study of men and women with documented myocardial infarction (MI) published in the paper “Low-density lipoprotein subclass patterns and risk of myocardial infarction,” Austin et al., J. Amer. Med. Assoc. 260: 1917-1921(4) (1988), it was reported that LDL phenotype B, the LDL subclass pattern characterized by a preponderance of small dense LDL particles, was associated with a 3-fold increased risk of MI. This association remained significant after adjustment for age, sex and relative weight. It has also been suggested that there may be a major genetic determinant for this LDL phenotype as in the paper “Inheritance of Low-density lipoprotein subclass patterns: results of complex segregation analysis,” Austin et al., Am J Hum Genet 73: 838-876 (5) (1988). Whether or not the relationship between LDL phenotype and CAD is independent of other risk factors such LDLc, HDLc or TRIG is still unclear.
High density lipoproteins (HDL) are responsible for the reverse transport of cholesterol from peripheral tissues back to the liver. Data from the paper “Altered particle size distribution of apoA-I-containing lipoproteins in subjects with coronary artery disease,” Cheung et al., J. Lipid Res. 32: 383-397 (1991), and the paper “Characterization of human high density lipoproteins by gradient gel electrophoresis,” Johansson et al, Biochim Biophys Acta 665: 708-719 (1991), would suggest that patients with documented CAD may have altered HDL particle size distribution when compared to that observed in non-CAD controls. In these studies, the heterogeneity of plasma HDL was assessed using a non-denaturing 4-30% polyacrylamide gradient gel first described in the paper “Characterization of human high density lipoproteins by gradient gel electrophoresis,” Blanche et al., Biochim Biophys Acta 665: 708-719 (1981).
A major impediment to large prospective studies of lipoprotein particle size distribution has been the unavailability of an efficient and reproducible method that can allow the determination of particle diameters for cholesterol-rich lipoproteins. This is mainly because high quality pre-cast gradient gels used in the earlier studies are no longer available commercially. The paper “Production of polyacrylamide gradient gels for the electrophoretic resolution of lipoproteins,” Rainwater et al., J. Lipid. Res. 33: 1876-1881 (1992), has reported a procedure for the preparation of a 4-30% gradient gel which provides estimates of HDL particle size comparable to those obtained with the PAA 4/30 gel (Pharmacia). In this gradient, however, LDL and larger lipoprotein particles tend to accumulate at the top of the gel, prohibiting the determination of particle size of these lipoproteins. A custom-made 2-16% gradient gel was also described by these investigators for the determination of LDL particle size in the paper “Effects of diabetes on lipoprotein size,” Singh et al., Arterioscl. Thromb. Vasc. Biol. 15: 1805-1811 (1995). Except for Gambert et al., who used lipid staining to visualize the LDL band as disclosed in the paper “Human low density lipoprotein fractions separated by gradient gel electrophoresis: Composition, distribution and alterations induced by cholesteryl ester transfer protein,” J. Lipid. Res. 31: 1199-1210 (1990), most investigators used Coomassie to stain the gels for protein after the electrophoresis. The use of a protein stain typically requires extensive staining and de-staining procedures for the gels after electrophoresis and special handling of the gels during these steps to maintain gel size and shape before scanning. Furthermore, by using a protein stain, many protein bands other than those corresponding to plasma lipoproteins are visible from the electrophoresis of whole plasma.
It is very difficult to make high quality of gradient gels for medical studies and clinic use. In the casting of the typical gradient gels, as shown in Rainwater et al. paper, the polyacrylamide solutions are commonly allowed to flow into a gel chamber from a stationary dispensing tip which is typically placed at the center of the gel. However, as the polyacrylamide solution flows from the dispensing tip to the sides of the plate, a secondary gradient is formed across the width of the gel resulting in lower gel concentrations toward the edges because of the diffusion of the solution. In order to reduce this diffusion effect, only narrow gels with 6-8 lanes across have been available to-date although a typical gel chamber is capable of having gels with up to 20 or more lanes. Moreover, uneven gradients and disturbances in the process of gel making due to the diffusion still exist even in the narrow gels.
SUMMARY OF THE INVENTION
Definitions
A number abbreviations used in this application for some frequently used technical terms are defined as the following:
The term “S-GGE” as used herein shall refer to a segmental gradient gel electrophoresis.
The term “S-GGE 2.8/8.30” as used herein shall refer to a 2.8/8.30 segmental gradient gel electrophoresis with a 2-8% gradient stacked above an 8-30% gradient.
The term “LIPOPROTEIN” as used herein shall refer to a class of plasma proteins that are complexed to lipids.
The term “TRIG” as used herein shall refer to triglycerides.
The term “CHOL” as used herein shall refer to cholesterol.
The term “LDL” as used herein shall refer to low density lipoproteins.
The term “HDL” as used herein shall refer to high density lipoproteins.
The term “Lp(a)” as used herein shall refer to lipoprotein(a) which consist of one LDL particle complexed to one apo(a) particle.
The term “LpB” as used herein shall refer to apob-containing lipoproteins.
The term “LDLc” as used herein shall refer to LDL-cholesterol.
The term “HDLc” as used herein shall refer to HDL-cholesterol.
The term “LpA-I” as used herein shall refer to apoA-I containing lipoproteins.
The term “LpA-I/A-II” as used herein shall refer to lipoproteins containing both apoA-I and apoA-II.
Summary
The present invention provides a new apparatus and method for making a uniform gel including a uniform, continuous gradient gel in many lanes occupying up to the capacity of a gel chamber. Moreover, the present invention can be practiced to produce a segmental gradient gel that would provide optimal conditions for the simultaneous characterization of LDL, Lp(a) and remnant lipoproteins (2-8% gradient) and HDL subclasses (8-30% gradient) from whole plasma. Additionally, the present invention allows the bands corresponding to all of the major lipid-carrying particles to be visualized without any handling of the gel. The present invention can also be practiced to make several gradient gels simultaneously. In sum, the present invention offers a new, better, and efficient gel making apparatus and method.
The present invention in one embodiment is a gel-making system that has a reservoir for holding a solution. The reservoir is connected to a movable arm through a tubing. The tubing has two ends: one end is in fluid communicatio

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